Pyocyanin and 1-Hydroxyphenazine Promote Anaerobic Killing of Pseudomonas aeruginosa via Single-Electron Transfer with Ferrous Iron

ABSTRACT Previously, it was reported that natural phenazines are able to support the anaerobic survival of Pseudomonas aeruginosa PA14 cells via electron shuttling, with electrodes poised as the terminal oxidants (Y. Wang, S. E. Kern, and D. K. Newman, J Bacteriol 192:365–369, 2010, https://doi.org/10.1128/JB.01188-09). The present study shows that both pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) promoted the anaerobic killing of PA14 Δphz cells presumably via a single-electron transfer reaction with ferrous iron. However, phenazine-1-carboxylic acid (PCA) did not affect anaerobic survival in the presence of ferrous iron. Anaerobic cell death was alleviated by the addition of antioxidant compounds, which inhibit electron transfer via DNA damage. Neither superoxide dismutase (SOD) nor catalase was able to alleviate P. aeruginosa cell death, ruling out the possibility of reactive oxygen species (ROS)-induced killing. Further, the phenazine degradation profile and the redox state-associated color changes suggested that phenazine radical intermediates are likely generated by single-electron transfer. In this study, we showed that the phenazines 1-OHPHZ and PYO anaerobically killed the cell via single-electron transfer with ferrous iron and that the killing might have resulted from phenazine radicals. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen which infects patients with burns, immunocompromised individuals, and in particular, the mucus that accumulates on the surface of the lung in cystic fibrosis (CF) patients. Phenazines as redox-active small molecules have been reported as important compounds for the control of cellular functions and virulence as well as anaerobic survival via electron shuttles. We show that both pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) generate phenazine radical intermediates via presumably single-electron transfer reaction with ferrous iron, leading to the anaerobic killing of Pseudomonas cells. The recA mutant defect in the DNA repair system was more sensitive to anaerobic conditions. Our results collectively suggest that both phenazines anaerobically kill cells via DNA damage during electron transfer with iron.


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Reviewer #2 (Comments for the Author)
The authors describe the ability of 1-OHPHZ and PYO to kill P. aeruginosa delta phz and provide evidence that these molecules are oxidized by ferrous iron that in turn damages bacterial DNA. The data support the conclusions in general.
[#1] Figure 6 of the manuscript is described vaguely and the figure needs additional labeling to follow. Is there another way to demonstrate the oxidized forms of these phenazines? mass spectroscopy?  As suggested by the Reviewer, we have added labels to Figure 6 for clarity ( Figure 6). As mentioned in the manuscript, phenazines are redox-active compounds with distinctive coloration, with two distinct peaks in the UV range and at least one in the visible range, according to the functional groups (Ref. #20 in the manuscript). In this study, we determined the redox states of both 1-OHPHZ and PYO by observing their colors to verify electron transfer between the phenazines and irons ( Figure 6). It is also known that oxidized form of PYO is bright blue color with a peak at 278 nm (Ref. #15 in the manuscript). Therefore, we measured UV spectra of oxidized PYO and 1-OHPHZ in the absence/presence of irons in the same conditions to confirm the observation (Supporting information 1). Consistent to the observation ( Figure 6B), the UV spectra showed that oxidized PYO (blue) has a peak at ~278 nm, whereas the peak was decreased in PYO partially reduced by ferrous iron (green) (Supporting information 1A). Similar to PYO, we also confirmed the redox state of 1-OHPHZ by both its colors and UV spectra (Supporting information 1B). The supporting information is not included in the manuscript because we believe that the colors of phenazines is sufficient to indicate their redox states. A B

Supporting information 1. UV spectra of oxidized PYO (A) and 1-OHPHZ (B) by adding irons in the absence of Pseudomonas cells under the anaerobic condition.
[#2] Does ferrous iron transfer electrons to 1-OHPHZ and PYO in other media? or are these observations specific to MOPS minimal medium?  In our study, we revealed that the phenazine intermediates are generated by electron transfer between both phenazines, 1-OHPHZ and PYO, and ferrous iron, and cause 3 anaerobic killing of PA14 ∆phz using MOPS medium. We agree with the Reviewer's suggestions regarding the use of other media to determine whether our observations are specific to MOPS media. MOPS is a Zwitterionic biological buffer and is frequently used in many biological and biochemical studies. It is best known as a useful buffering agent for RNA isolation and protein purification (1). Moreover, MOPS is also used as a non-coordinating buffer for solutions containing metal ions because of lack of the ability to form chelates with most metal ions (1). Autoxidation of transitional metal ions, such as ferrous iron, depends on the reaction conditions, and the rate varies in the presence of different anions. Many studies on metal autoxidation have been performed using unbuffered solutions or different buffers adjusted to various pH. Buffers can affect metal autoxidation (2,3). Tadolini (1987) reported that ferrous iron is substantially stable for significant time in Mops buffer compared to other buffers. For the reasons, it is widely used in biochemical studies, especially in the study of the redox state of phenazine or iron (4)(5)(6)(7)(8)(9)(10)(11). We believe that MOPS medium is most suitable for studying the redox state by electron transfer excluding other influencing factors.
[#3] DNA damage can be measured. Some other assay is needed to confirm that DNA damage has occurred in the PYO and/or 1-OHPHZ and Fe(II) exposed cells besides CFU decrease.  We fully agree with the Reviewer's suggestion that DNA damage caused by the phenazine intermediates should be directly measured for definitive conclusions. In this study, we demonstrated that the phenazine intermediates induces DNA damage by measuring the CFU of the recA mutant ( Figure 8). Since RecA is important for DNA repair in bacteria (Ref. #44), the recA mutants are widely used to assess DNA damage (12)(13)(14)(15). Thus, we believe that the CFU data of the mutant is sufficient to clearly show that the intermediates cause DNA damage. In our ongoing follow-up study, we performed a random transposon mutagenesis of PA14 ∆phz to elucidate the mechanism of the anaerobic killing. We screened Tn mutants which are resistant to the anaerobic killing condition, compared to a parental strain. Among them, a mutant carries a Tn5 transposon insertion in a putative ndh gene encoding NADH dehydrogenase.

PYO 1-OHPHZ A B
Supporting information 2. Anaerobic killing of PA14 ∆phz and a Tn mutant by PYO (A) or 1-OHPHZ (B) via electron transfer with ferrous iron. 4 CFU data show that the Tn mutant is resistant to the phenazine intermediates (Supporting information 2, unpublished data) as well as two antibiotics, ciprofloxacin and norfloxacin (Supporting information 3, unpublished data). These antibiotics belonging to the class of fluoroquinolone, induce DNA damage by inhibiting DNA gyrase and topoisomerase IV (16,17). Therefore, our current data support that the phenazine intermediates induce DNA damages (Supporting information 2 & 3, unpublished data).

Supporting information 3.
Cell viability of PA14 ∆phz and a Tn mutant in the presence of antibiotics.